The human immunodeficiency virus (HIV) is believed to effect the development and progress of AIDS through infection of CD4.sup.+ T cells, macrophage and other lymphoid and nonlymphoid cell types that contain this marker. The population of CD4.sup.+ cells is generally used as a measure of AIDS progression, since CD4.sup.+ cells are continuously depleted in the course of the disease. The mechanism of CD4.sup.+ cell depletion has not been elucidated; however, the major cytolytic effect of HIV infection on CD4.sup.+ cells in culture is an envelope-mediated syncytium induction. Other mechanisms which may be effective in vivo include stimulation of the production of cytokines, clearance of HIV-infected cells by cytotoxic lymphocytes and autoimmune mechanisms, and apoptosis.
Based on general concepts of the mechanism of retroviral infection and the nature of the infected cell, three types of drugs to arrest the progress of AIDS have been proposed. One class contains those which block reverse transcription such as azidothymidine (AZT), another class comprises protease inhibitors that prevent maturation of the virions, and the third class comprises drugs such as soluble CD4 which prevent interaction between the virion and the CD4 receptor. The efficacy of each of these classes of drugs is limited, and there is clearly a need for more effective ways to arrest the progress of this condition.
The approach of the present invention takes advantage of the critical role of the HIV-encoded protein Vpr. Vpr is one of nine proteins known to be encoded by the HIV genome. A diagram of the HIV genome, shown schematically, is set forth in FIG. 1. Vpr was initially identified as an open reading frame in the HIV genome. Expression of this open reading frame was confirmed by the demonstration that individuals infected with HIV develop antibodies against the Vpr gene product (Gras-Masse, H. et al. Int J Pept Prot Res (1990) 36:219; Reiss, P. et al. J AIDS (1990) 3:115. The gene product was shown to be weak transcriptional activator by Cohen, E. A. et al. J AIDS (1990) 3:11-18 and by Ogawa, K. et al. J Virol (1990) 63:4110-4114. The protein appears to be present in virions at an equimolar amount compared to the major gag-encoded capsid proteins and has been shown to interact with the gag-encoded protein p6. Cohen, E. A. et al. J Virol (1990) 64:3097-3099, Yu, et al. J Virol (1990) 64:5688; Yuan, X. et al. AIDS Res Cum Retro (1990) 6:1265; Paxton, W. et al. J Virol (1993) 67:7229; Lavallee, C. et al. J Virol (1994) 68:1926. For interaction with p6, the carboxy terminal amino acids at positions 84-94 are required. Paxton, W. et al. J Virol (supra).
The human HIV Type 2 Vpr gene has been shown as essential for productive infection of human macrophage by Hattori, N. et al. Proc Natl Acad Sci USA (1990) 87:8080-8084; however, Balotta, C. J Virol (1993) 67:4409 has shown Vpr to be unnecessary for replication of the virus in immortalized T cell lines or peripheral blood lymphocytes. The Vpr protein has been shown to provide a mechanism for nuclear localization of viral nucleic acids in nondividing cells by Heinzinger, N. K. et al. Proc Natl Acad Sci USA (1994) 91:7311-7315. In addition, Lang, S. M. et al. J Virol (1993) 67:902-912 have shown that mutations in Vpr attenuate the pathogenicity of macaques infected with SIV. Further, Tristem, M. et al. EMBO J (1992) 11:3405 showed that Vpr and Vpx are highly conserved among primate lentiviruses. Levy, D. N. et al. Cell (1993) 72:541 demonstrated that Vpr induces differentiation and growth arrest in human rhabdomyosarcoma cells.
It is therefore known that Vpr is not a null protein with respect to infection by HIV. However, the central role played by this protein in permitting virus to multiply while effecting depletion of the very cells which are infected by the virus has not been appreciated. The present applicants have demonstrated that Vpr arrests the development of cells in which it is contained at the G2 stage of the life cycle. Arrest at this particular point is significant since HIV integration occurs during the previous S phase of the cell cycle. Also, preventing the cells from entering the subsequent mitotic stage (M) prevents infected cells from entering the GO stage in which the relative metabolic inactivity of the cells would be unfavorable for adequate viral production. Furthermore, arrest of cells in the G2 stage may prevent apoptosis thus, also, permitting continued viral production. Importantly, it also prevents T cell clonal expansion.
The Vpr protein and its interaction with intracellular targets in the infected cell are crucial to the success of the infective virus. Therefore, therapeutic agents which interrupt this interaction will also successfully arrest the progress of the disease.